Calcium levels in the PG vary during the course of the day.

<p>(A) <i>Left</i>: Representative images of GCaMP fluorescence in the PG before (t = 0), and 10 min and 30 min after incubation with 50μM calcimycin (external [calcium]: 1mM) and <i>Right</i>: quantitation of the fluorescence in the PG; numbers in parenthesis indicate number of PG glands measured; *<i>p</i><0.05 (paired <i>t</i>-test compared with signal measured before adding drug). (B) Dose response of GCaMP sensor measured using increasing extracellular calcium concentrations; n: number of PG cells (taken from at least 4 PGs per condition). (C) Levels of fluorescence in controls and in animals bearing knockdown of <i>calcium channels</i> (<i>cacophony</i> [<i>cac</i>] and <i>Ca-alpha1D</i>), <i>IP3</i> receptor (<i>IP3R</i>) and <i>SERCA</i> in the PG (using <i>phm</i>-GAL4 driver)(all measured at ZT12). The number of cells analyzed is indicated above colored circle. For each experiment 4–8 animals were examined. Error bars denote SEM. (D-G) Levels of fluorescence in the PG of wildtype animals at different times of day under LD (D) and DD (E) conditions, and in the PG of <i>per</i>⁰ mutants under LD (F) and DD (G) conditions. Different letters indicate statistically significant differences (ANOVA with Tukey’s <i>post hoc</i> test). The number of cells is indicated in brackets and above each error bars. In A: PG: prothoracic gland, CA: <i>corpus allatum</i>, RG: ring gland. Scale bar 10μm; in A-G: Normalized fluorescence is quantitated in arbitrary units.